流式微球联合检测炎症性因子IL-6、TNF-α和MCP-1方法学的建立和评价令狐颖1张程2陈艳2黄山1*许健1刘志琴3（1.贵州省人民医院临床检验中心，贵州贵阳550002；2.贵阳医学院09级硕士研究生；3.本院心内科）【摘要】目的建立流式微球分析技术联合检测人血清中细胞介素-6（IL-6）、肿瘤坏死因子-α（TNF-α）和单核细胞趋化因子-1（MCP-1）的方法并对其进行系统性评价。方法分别将三种选择好一定浓度的捕获抗体（IL-6、TNF-α和MCP-1鼠抗人单克隆抗体）包被在三种已激活的不同荧光强度编码的羧基化聚苯乙烯微球上，用正交试验对试验条件进行优化选择，并应用CLSI的有关规则进行方法学评价。结果实验选择各种捕获单克隆抗体的加入量为10µg，各种生物素标记的羊抗人单克隆抗体的稀释倍数为1:540，第一次反应时间为2h、洗涤二次，PE标记亲和素的稀释倍数为1:1000，第二次反应1h洗涤一次。方法学评价显色：IL-6线性范围是1.13—3333.33pg/mL，批内变异系数为1.98%—2.31%，批间变异系数为3.52%—4.17%，准确度相对偏倚为0.19%—0.66%，回收率是95.4—101.5%，灵敏度为1.13pg/mL；TNF-α线性范围是1.07—1111.11pg/mL，批内变异系数为2.67%—2.90%，批间变异系数为4.27%—4.91%，准确度相对偏倚为0.85%—1.53%，回收率是97.3—104.2%，灵敏度为1.07pg/mL；MCP-1线性范围是7.07—3333.33pg/mL，批内变异系数为2.63%—2.92%，批间变异系数为3.70%—4.23%，准确度相对偏倚为0.15%—1.06%，回收率是95.2—104.3%，灵敏度为7.07pg/mL.高浓度的甘油三酯、胆固醇和胆红素对三种因子有一定的干扰率，低浓度的甘油三酯、胆固醇和胆红素对三种因子干扰较小。分别与ELISA方法比较无显著差异结论自建的IL-6、TNF-α和MCP-1流式微球联合检测技术，可拓展流式细胞分析技术，值得临床推广使用。【关键词】流式微球分析技术（CBA）；联合检测；细胞介素-6（IL-6）；肿瘤坏死因子-α（TNF-α）；单核细胞趋化因子-1（MCP-1）)DevelopmentandevaluationofaCytometricBeadAssayMultiplexDetectionMethodforthedetectionofIL-6、TNF-αandMCP-1LING-Huying1,HUANGShan1，ZHANGCheng2,CHENYan2,XUJian1，LIUZhi-qin3(1.GuizhouProvinceCenterforClinicalLaboratory;2.GuiyangMedicalCollege;3.GuizhouProvincePeople'sHospital)1基金项目：贵阳市科技局社会发展领域科技攻关项目[2010]筑科农合同字第1-社-35号和贵州省卫生厅立项资助项目[Gzwlj2009-1-007]。*通讯作者。【Abstract】ObjectiveTodevelopmentandSystematicreviewofacytometricbeadarrayMultiplexDetectionMethodforthedetectionofIL-6,TNF-αandMCP-1.MethodAcertainconcentrationofthreekindsofcaptureantibody(mouseanti-humanIL-6monoclonalantibody,mouseanti-humanTNF-αmonoclonalantibody,mouseanti-humanMCP-1monoclonalantibody)wererespectivelycoatedwiththreekindsofactivatedbeads,andaccordingorthogonalexperimenttoselectthebesttestconditions,andapplyingtherelevantrulesofCLSItomethodologyevaluation.ResultInthereaction,thebestquantityofthethreekindsofmouseanti-humanmonoclonalantibodywas10μg,thebestdilutionofbiotin-labeledmonoclonalantibodywas1:540,thebestincubationtimewas2hours,thefirstwashingtimewas2,thebeatdilutionofstreptavidin-PEwas1:1000,thebestincubationtimeofstreptavidin-PEwas1hours,thesecondwashingtimewas1.Accordingthemethodologyevaluation,thelinearrangeofIL-6was1.13-3333.33pg/ml,theintra-assayofvariationwas1.98%and2.31%,theinter-assayofvariationwas3.52%and4.17%,therelativebiaswas0.19%and0.66%,therecoverywas95.4-101.5%,thesensitivitywas1.13pg/ml;thelinearrangeofTNF-αwas1.07-1111.11pg/ml,theintra-assayofvariationwas2.67%and2.90%,theinter-assayofvariationwas4.27%and4.91%,therelativebiaswas0.85%and1.53%,therecoverywas97.3-104.2%,thesensitivitywas1.07pg/ml;thelinearrangeofMCP-1was7.07-3333.33pg/ml,theintra-assayofvariationwas2.63%and2.92%...
